Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress

Abstract

Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (K1, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.